1. Field of the Invention
The present invention relates to a reversible fluorescence photoswitch based on the dye-crosslinked dendritic nanoclusters for high-contrast imaging of living biological systems.
2. Description of the Related Art
Since Denkewalter synthesized the polylysine dendrimer for the first time in the late 1970s, research on dendrimers has been conducted actively, which includes design of new dendrimer structures and development of efficient synthetic strategies, understanding physicochemical and structural properties of various types of dendrimers and utilization of those information in building dendrimer-based highly ordered structures (e.g., self-assembly) and biomimetic systems, and applications of dendrimers in materials sciences and biomedicine. The following is an example of the structure of polylysine dendrimer synthesized by Denkewalter [U.S. Pat. No. 4,410,688].

Dendrimers are relatively small (ca. <10 nm in diameter) tree-like (radial-shaped) unimolecular polymers which are made by stepwise iterative synthesis. Unlike other conventional polymers, the size of a dendrimer is somewhat predictable and robust in a specific environment (solvent, pH, temperature, etc.). Therefore, dendrimers are in a highly favorable position because they can be customized to various applications, particularly those which are sensitive to the hydrodynamic diameters. Specifically, the advantages of dendrimers include structural integrity, possibility to control the component functional groups and their corresponding physical properties by chemical synthesis, feasibility to conjugate multiple functional units (small-molecule drugs, targeting units, surface modifiers, etc.) at the periphery and the interior, and a low enzymatic degradation rate.
Applications of dendrimers in biomedical research include the usage of polycationic dendrimers to form charge complexes with negatively charged genes for efficient gene transfection, the usage of dendrimers as delivery vehicle to either physically encapsulate or covalently attach the small-molecule drugs for their controlled release at the diseased sites in response to specific stimuli (pH, light, enzyme, etc.), the structural modification of dendrimer scaffolds for targeted delivery or release of drugs at a controlled rate, the enhancement of binding affinity in the ligand-receptor interactions at the extracellular matrix through the multivalent effect, attachment of multiple copies of imaging agents at the dendrimer scaffolds to facilitate diagnosis through signal amplification, and the artificial tissue engineering using biocompatible and/or biodegradable dendrimers.
Among many types of dendrimers, poly(amidoamine) (hereinafter, referred to as “PAMAM”) dendrimer was developed by Dr. Donald A. Tomalia while he was at the Dow Chemical in the 1980s. The interior of the commercial PAMAM dendrimers is composed of aliphatic amine and amide groups and their surface groups can be amine, carboxylic acid, or hydroxyl groups. The following structures illustrate PAMAM dendrimers with the ethylenediamine as a core unit and the amine as terminal groups (A: second generation (G2) PAMAM dendrimer; B: G3 PAMAM dendrimer).

Photochromism refers to the phenomenon where the color of a compound or a system containing such compound changes reversibly by irradiation with UV and visible light. Photochromic compounds interconvert between two or more isomeric forms with different absorption properties and refractivity by irradiation using the light with its wavelength at the absorption range of each isomer.
Photochromic materials which can change color reversibly by light have applicability in various fields such as photorecorders, photoswitches, modulators, and the like. For example, diarylethene derivatives are photochromic compounds which change their colors upon exposure to UV light and revert to their original colors when irradiated with visible light. These diarylethene derivatives were first synthesized in 1985, and have been known as thermally stable photochromic compounds. Various diarylethene derivatives have been synthesized to date, and those substituted with fluorine are known to be particularly stable and rapid in the reversible color change [M. Irie, Chem. Rev. 2000, 100, 1685-1716; S. Nakamura, et al., J. Photochem. Photobiol. A: Chem. 2008, 200, 10-18].
While diarylethene derivatives dissolve well in most of the organic solvents, the use of diarylethene derivatives for biological applications has been limited due to their significantly low water-solubility. A recent report showed derivatization of diarylethene compounds with oligo(ethylene glycol) groups to convert them to water-soluble compounds [T. Hirose, et al., J. Org. Chem. 2006, 71, 7499-7508]. In addition, Japanese Patent Publication No. 2003-246776 discloses a method of crosslinking a photochromic compound and a biomolecule with a thiol functional group using the maleimide derivative in order to provide crosslinkable photochromic molecules which reversibly switched the structure of a biofunctional molecule and to provide a compound that can induce the biofunctional molecule capable of producing the mechanical energy. However, such molecules were inappropriate for biological applications due to their insolubility in water.
Photochromic compounds show reversible color change by irradiation with the light of a specific wavelength, which can be detected easily with UV-Vis spectrophotometer. As such, it may be useful to develop a detection method for biomolecules based on photochromic compounds that will allow for the reduction of the undesired background signals and improve the detection sensitivity.
Photochromic FRET (pcFRET) refers to the reversible switching of the fluorescence of a neighboring fluorophore through the fluorescence resonance energy transfer (FRET) using photochromic compounds such as azobenzene, diarylethene, spiropyran, fulgide, and so on as switch molecules [M. Irie et al., Nature 2002, 420, 759-760; L. Giordano, et al., J. Am. Chem. Soc. 2002, 124, 7481-7489; N. Soh, et al., Chem. Commun. 2007, 5206-5208]. A few recent examples exhibited applications of pcFRET to living biological systems for their reversible fluorescence switching [Y. Zou, et al., J. Am. Chem. Soc. 2008, 130, 15750-15751; U. Al-Atar, et al., J. Am. Chem. Soc. 2009, 131, 15966-15967; A. A. Beharry, et al., Angew. Chem. Int. Ed. 2011, 50, 1325-1327]. The most suitable photochromic compound for pcFRET is diarylethene because diarylethene derivatives generally have high thermal stability, high fatigue resistance, high sensitivity, and rapid response time.
The present inventors developed dendritic nanoclusters by oligomerizing dendrimers using a photochromic compound as a crosslinker where covalent linkages were formed between the surface groups of the dendrimers and the end groups of the photochromic compound and subsequently, by attaching a fluorophore and the excess amount of biocompatible surface modifiers to the surface of the dendrimer. These dendritic nanoclusters greatly improved the detection sensitivity of the fluorescence inside the biological systems and had high biocompatibility, and thereby the present inventors completed the present invention.